Isolated, MAGE-3 derived peptides which complex with HLA-A2 molecules and uses thereof

ABSTRACT

Tumor rejection antigens derived from tumor rejection precursor MAGE-3 have been identified. These &#34;TRAS&#34; bind to the MHC-class I molecule HLA-A2, and the resulting complexes stimulate the production of cytolytic T cell clones which lyse the presenting cells. The peptides and complexes may be used diagnostically, therapeutically, and as immunogens for the production of antibodies, or as targets for the generation of cytolytic T cell clones.

RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/217,186, filed Mar. 24, 1994 now U.S. Pat. No. 5,585,461.

FIELD OF THE INVENTION

This invention relates to immunogenetics and to peptide chemistry. Moreparticularly, it relates to peptides, especially deca- and nonapeptidesuseful in various ways, including immunogens and as ligands for HLA-A2molecules. More particularly, it relates to a so-called "tumor rejectionantigen", derived from the tumor rejection antigen precursor encoded bygene MAGE-3, and presented by the MHC-class I molecule HLA-A2.

BACKGROUND AND PRIOR ART

The study of the recognition or lack of recognition of cancer cells by ahost organism has proceeded in many different directions. Understandingof the field presumes some understanding of both basic immunology andoncology.

Early research on mouse tumors revealed that these displayed moleculeswhich led to rejection of tumor cells when transplanted into syngeneicanimals. These molecules are "recognized" by T-cells in the recipientanimal, and provoke a cytolytic T-cell response with lysis of thetransplanted cells. This evidence was first obtained with tumors inducedin vitro by chemical carcinogens, such as methylcholanthrene. Theantigens expressed by the tumors and which elicited the T-cell responsewere found to be different for each tumor. See Prehn, et al., J. Natl.Canc. Inst. 18:769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572(1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res.30:2458-2462 (1970) for general teachings on inducing tumors withchemical carcinogens and differences in cell surface antigens. Thisclass of antigens has come to be known as "tumor specifictransplantation antigens" or "TSTAs". Following the observation of thepresentation of such antigens when induced by chemical carcinogens,similar results were obtained when tumors were induced in vitro viaultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53:333-1336(1974).

While T-cell mediated immune responses were observed for the types oftumor described supra, spontaneous tumors were thought to be generallynon-immunogenic. These were therefore believed not to present antigenswhich provoked a response to the tumor in the tumor carrying subject.See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).

The family of tum⁻ antigen presenting cell lines are immunogenicvariants obtained by mutagenesis of mouse tumor cells or cell lines, asdescribed by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), thedisclosure of which is incorporated by reference. To elaborate, tum⁻antigens are obtained by mutating tumor cells which do not generate animmune response in syngeneic mice and will form tumors (i.e., "tum⁺ "cells). When these tum⁺ cells are mutagenized, they are rejected bysyngeneic mice, and fail to form tumors (thus "tum⁻ "). See Boon et al.,Proc. Natl. Acad. Sci. USA 74:272 (1977), the disclosure of which isincorporated by reference. Many tumor types have been shown to exhibitthis phenomenon. See, e.g., Frost et al., Cancer Res. 43:125 (1983).

It appears that tum⁻ variants fail to form progressive tumors becausethey initiate an immune rejection process. The evidence in favor of thishypothesis includes the ability of "tum⁻ " variants of tumors, i.e.,those which do not normally form tumors, to do so in mice with immunesystems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl.Acad. Sci. USA 76: 5282-5285 (1979); and the observation thatintraperitoneally injected tum⁻ cells of mastocytoma P815 multiplyexponentially for 12-15 days, and then are eliminated in only a few daysin the midst of an influx of lymphocytes and macrophages (Uyttenhove etal., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes theobservation that mice acquire an immune memory which permits them toresist subsequent challenge to the same tum⁻ variant, even whenimmunosuppressive amounts of radiation are administered with thefollowing challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA74:272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra).

Later research found that when spontaneous tumors were subjected tomutagenesis, immunogenic variants were produced which did generate aresponse. Indeed, these variants were able to elicit an immuneprotective response against the original tumor. See Van Pel et al., J.Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it ispossible to elicit presentation of a so-called "tumor rejection antigen"in a tumor which is a target for a syngeneic rejection response. Similarresults have been obtained when foreign genes have been transfected intospontaneous tumors. See Fearon et al., Cancer Res. 48: 2975-1980 (1988)in this regard.

A class of antigens has been recognized which are presented on thesurface of tumor cells and are recognized by cytolytic T cells, leadingto lysis. This class of antigens will be referred to as "tumor rejectionantigens" or "TRAs" hereafter. TRAs may or may not elicit antibodyresponses. The extent to which these antigens have been studied, hasbeen via cytolytic T cell characterization studies, in vitro i.e., thestudy of the identification of the antigen by a particular cytolytic Tcell ("CTL" hereafter) subset. The subset proliferates upon recognitionof the presented tumor rejection antigen, and the cells presenting theantigen are lysed. Characterization studies have identified CTL cloneswhich specifically lyse cells expressing the antigens. Examples of thiswork may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boonet al., J. Exp. Med. 152:1184-1193 (1980); Brunner et al., J. Immunol.124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634(1980); Maryanski et al., Eur. J. Immunol. 126: 406-412 (1982);Palladino et al., Canc. Res. 47: 5074-5079 (1987). This type of analysisis required for other types of antigens recognized by CTLs, includingminor histocompatibility antigens, the male specific H-Y antigens, andthe class of antigens referred to as "tum-" antigens, and discussedherein.

A tumor exemplary of the subject matter described supra is known asP815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278(1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al.,J. Exp. Med. 172: 35-45 (1990), the disclosures of which areincorporated by reference. The P815 tumor is a mastocytoma, induced in aDBA/2 mouse with methylcholanthrene and cultured as both an in vitrotumor and a cell line. The P815 line has generated many tum⁻ variantsfollowing mutagenesis, including variants referred to as P91A (DePlaen,supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast totumor rejection antigens--and this is a key distinction--the tum⁻antigens are only present after the tumor cells are mutagenized. Tumorrejection antigens are present on cells of a given tumor withoutmutagenesis. Hence, with reference to the literature, a cell line can betum⁺, such as the line referred to as "P1", and can be provoked toproduce tum⁻ variants. Since the tum⁻ phenotype differs from that of theparent cell line, one expects a difference in the DNA of tum⁻ cell linesas compared to their tum⁺ parental lines, and this difference can beexploited to locate the gene of interest in tum⁻ cells. As a result, itwas found that genes of tum⁻ variants such as P91A, 35B and P198 differfrom their normal alleles by point mutations in the coding regions ofthe gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58:293-303 (1989). This has proved not to be the case with the TRAs of thisinvention. These papers also demonstrated that peptides derived from thetum⁻ antigen are presented by the L^(d) molecule for recognition byCTLs. P91A is presented by L^(d), P35 by D^(d) and P198 by K^(d).

PCT application PCT/US92/04354, filed on May 22, 1992 assigned to thesame assignee as the subject application, teaches a family of humantumor rejection antigen precursor coding genes, referred to as the MAGEfamily. Several of these genes are also discussed in van der Bruggen etal., Science 254: 1643 (1991). It is now clear that the various genes ofthe MAGE family are expressed in tumor cells, and can serve as markersfor the diagnosis of such tumors, as well as for other purposesdiscussed therein. See also Traversari et al., Immunogenetics 35: 145(1992); van der Bruggen et al., Science 254: 1643 (1991). The mechanismby which a protein is processed and presented on a cell surface has nowbeen fairly well documented. A cursory review of the development of thefield may be found in Barinaga, "Getting Some `Backbone`: How MHC BindsPeptides", Science 257: 880 (1992); also, see Fremont et al., Science257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron etal., Science 257: 964 (1992). These papers generally point to arequirement that the peptide which binds to an MHC/HLA molecule be nineamino acids long (a "nonapeptide"), and to the importance of the firstand ninth residues of the nonapeptide. As described herein, while this"rule" is generally true, there is some leeway as to the length ofpeptides which MHC-class I molecules will bind.

Studies on the MAGE family of genes have now revealed that a particularnonapeptide is in fact presented on the surface of some tumor cells, andthat the presentation of the requires that the presenting molecule beHLA-A1. Complexes of the MAGE-1 tumor rejection antigen (the "TRA" ornonapeptide") leads to lysis of the cell presenting it by cytolytic Tcells ("CTLs").

Attention is drawn, e.g., to concurrently filed application Serial No.08/217,187 to Traversari et al., and Ser. No. 08/217,188 to Melief etal., both of which present work on other, MAGE-derived peptides.

Research presented in, e.g., U.S. patent application Ser. No. 07/938,334filed Aug. 31, 1992, and in U.S. patent application Ser. No. 073,103,filed Jun. 7, 1993, showed that when comparing homologous regions ofvarious MAGE genes to the region of the MAGE-1 gene coding for therelevant nonapeptide, there is a great deal of homology. Indeed, theseobservations lead to one of the aspects of the invention disclosed andclaimed therein, which is a family of nonapeptides all of which have thesame N-terminal and C-terminal amino acids. These nonapeptides weredescribed as being useful for various purposes which includes their useas immunogens, either alone or coupled to carrier peptides. Nonapeptidesare of sufficient size to constitute an antigenic epitope, and theantibodies generated thereto were described as being useful foridentifying the nonapeptide, either as it exists alone, or as part of alarger polypeptide.

These references, especially Ser. No. 073,103, showed a connectionbetween HLA-A1 and MAGE-3; however, only about 26% of the caucasianpopulation and 17% of the negroid population presents HLA-A1 moleculeson cell surfaces. Thus, it would be useful to have additionalinformation on peptides presented by other types of MHC molecules, sothat appropriate portions of the population may benefit from theresearch discussed supra.

It has now been found that antigen presentation of MAGE-3 derivedpeptides is not limited to HLA-A1 molecules. The invention set forth, inthe disclosure which follows, identifies peptides which complex with MHCclass I molecule HLA-A2. The ramifications of this discovery, whichinclude therapeutic and diagnostic uses, are among the subjects of theinvention, set forth in the disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the results of initial screening data on the peptidesdescribed herein.

FIG. 2 shows titration data obtained using SEQ ID NO: 2 and SEQ ID NO:6.

FIG. 3 depicts results obtained in experiments designed to determinewhether CTLs specific for complexes of peptide and HLA molecule could beprovoked. The X axis shows ratio of effector target cells, while the Yaxis shows percent lysis, via chromium release.

FIGS. 4A, 4B, 4C and 4D show the percentage of lysis obtained with eachof four specific CTLs found as a result of limiting dilution assays. Thetargets were T2 cells.

FIGS. 5A-5D show results obtained with each of the CTLs when testedagainst a variety of transformed and non-transformed cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

The methodologies employed in this set of experiments are akin to thosedescribed by Elvin et al., J. Imm. Meth. 158: 161-171 (1993), Townsendet al., Nature 340:443-448 (Aug. 10, 1989), and Townsend et al., Cell62:285-290 (Jul. 27, 1990), all of which are incorporated by referencein their entirety.

Cell line 0.174 as described was used. It is an HLA-A2 presenting cellline deficient in the pathway which supplies peptides to the endoplasmicreticulum, the site of assembly of MHC class-I heterodimers. The cellline can assemble MHC class-I molecules, but these are unstable and, oncell lysis, dissociate into free heavy and light chains during overnightincubation. The heterodimers can, however, be stabilized in vitro viaaddition of appropriate peptide ligands. (Townsend et al., Nature 340:443-448 (1989); Townsend et al., Cell 62: 285-295 (1990)). Thus, thestabilized molecules can be immunoprecipitated with antibodies specificfor the MHC class-I molecule.

In the first part of these experiments, peptides were tested todetermine if they facilitated assembly of HLA-A2 in the cell line. Thepeptides tested included the following:

SEQ ID NO: 1 Gly Leu Glu Ala Arg Gly Glu Ala Leu

SEQ ID NO: 2 Ala Leu Ser Arg Lys Val Ala Glu Leu

SEQ ID NO: 3 Cys Leu Gly Leu Ser Tyr Asp Gly Leu

SEQ ID NO: 4 Ile Leu Gly Asp Pro Lys Lys Leu Leu

SEQ ID NO: 5 His Leu Try Ile Phe Ala Thr Cys Leu

SEQ ID NO: 6 Phe Leu Trp Gly Pro Arg Ala Leu Val

SEQ ID NO: 7 Thr Leu Val Glu Val Thr Leu Gly Glu Val

SEQ ID NO: 8 Ala Leu Ser Arg Lys Val Ala Glu Leu Val

SEQ ID NO: 9 Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val

SEQ ID NO: 10 Ala Leu Val Glu Thr Ser Yr Val Lys Val

Cells were labelled by exposure to [³⁵ S] methionine (aliquots of1--2×10⁷ cells, labeled with 100-200 μCi, 60 minutes of contact). Thecells were then washed, once, with phosphate buffered saline, and thenresuspended in 10 ml of lysis buffer (0.5% NP-40; 0.5% Mega 9, 150 mMNaCl, 5 mM EDTA, 50 mM Tris [pH 7.5], 2 mM phenylmethylsulfonylflouride,5 mM iodoacetamide). The lysates were then incubated with peptide (10 μMand 20 μM), for 15-18 hours. Nuclei were then pelleted in a microfuge,and the lysates were precleared, overnight, at 4° C. with 0.2 ml ofwashed, 10% (w/v) Staphylococcus A organisms. Lysates were divided intotwo portions, and monoclonal antibody BB7.2 was added to a finalconcentration of 5 ug/ml. This mAb is a conformation specific, HLA-A2recognizing mAb described by Parham et al., Hum. Immunol. 3: 277-299(1981). The mixtures were incubated for 90 minutes on ice, followed byaddition of bovine serum albumin to 1% (w/v), and 100 ul of 5% (w/v)protein-A Sepharose beads. Tubes were rotated for 45 minutes, afterwhich beads were washed, four times, with 1 ml wash buffer (0.5% NP-40,150 mM NaCl, 5 mM EDTA, 50 mM Tris [PH 7.5]). Samples were eluted, andanalyzed on 12% polyacrylamide gels, in accordance with Townsend et al.,Nature 340: 443-448 (1989) .

FIG. 1 shows results from these experiments for the peptides which gavepositive results. These were SEQ ID NOS: 2, 6, 7, 8 and 10, as isevidenced by the dark band, indicated by HC (heavy chain) common to allof the gels, and represents immunoprecipitated MHC molecule (HLA-A2)that had complexed with the peptide prior to electrophoresis.

The figure shows work with SEQ ID NOS: 2, 6, 7, 8 and 10, running fromleft to right. The vertical bar separates SEQ ID NO: 5 from gels marked"0.174", "A2 line", and "0.174 matrix". 0.174 is a "negative" controlfor the heavy chain of the MHC class I molecule. As noted supra, thiscell line does not present stable MHC-class I molecules withoutexogenous peptide, and as mAb BB7.2 is conformation specific, it shouldnot precipitate uncomplexed MHC-class I molecules. "A2" refers to aknown cell line presenting HLA-A2 (the line is LBL 721, described byDeMars et al., Hum. Immunol. 11: 77 (1984)), but any cell presentingstable HLA-A2 molecules would function in the same way. "0.174 matrix"shows results when 0.174 cell line was incubated together with thecontrol peptide GILGFVFTL (SEQ ID NO: 11), which is derived frominfluenza virus and is known to be presented by HLA-A2.

The results show the stabilization of the MHC-class I molecule, by thefact that the bands for "HC" (heavy chain) are comparable to thoseobtained for A2 and 0.174 matrix. In fact, the MHC molecule is disruptedby the reducing gel; however, the heavy chain molecules will be bound bythe comformation specific mAb if stabilized prior to reduction. This isin fact what the gels show --i.e.--that the recited peptides bound tothe HLA-A2 molecules, and stabilized them.

EXAMPLE 2

Once binding peptides were identified, a series of titration experimentswere carried out. In these, varying concentrations of peptides, inaccordance with Townsend et al., Cell 62: 285-295 (Jul. 27, 1990) at293, incorporated by reference herein, were added to lysates of the cellline referred to supra, and immunoprecipitated to determine theconcentration which was the best concentration for the binding of thepeptide.

FIG. 2 shows the results obtained for two of the peptides i.e., SEQ IDNO: 2 and 6. The peptides were titrated against a known HLA-A2 bindingpeptide SEQ ID NO: 11, with 10 fold dilutions starting at 20 μM, anddecreasing to 2, 0.2 and 0.002 μM.

Experiments were carried out with these peptides (i.e., SEQ ID NOS: 2and 6). In the case of SEQ ID NO: 2, in experiments not reported herethe peptide titrated at 5-10 nM. This was comparable to the control (SEQID NO: 11).

EXAMPLE 3

A series of experiments were carried out to show the ability of thepeptide SEQ ID NO: 6 to provoke lysis by cytolytic T lymphocytes("CTLs") specific to complexes of the peptide and HLA-A2. The firststeps in these experiments are described herein.

Peripheral blood lymphocytes ("PBLs") were taken from a normal donor,i.e., one without any cancer tumors. The donor, referred to as "LB705"was typed as HLA-A1, A2, B8, B27. At the start ("day 0"), PBLs from thedonor were suspended, at 10⁶ cells/ml, in Iscove's medium and 10% fetalcalf serum and "AAG" (asparagine+arginine+glutamine), and 20 ug/ml ofrabbit antihuman IgM antibody, and 20 ng/ml recombinant human IL-4("r-hu-IL4"), and 0.005% Pansorbin cells. The mixture was distributedinto 24-well tissue culture plates (2 ml per well).

At day 3, the cells were centrifuged and resuspended in Iscove's mediumand 10% human serum and AAG and 20 ng/ml r-hu-IL4.

Two days later, on day 5, the cells were again centrifuged, andresuspended in fresh Iscove's medium and 10% human serum and AAG and 20ng/ml r-hu-IL-4, and 20 U/ml recombinant human γ-interferon.

On day 6, the cells were again centrifuged, and resuspended at 5×10⁶cells/ml in Iscove's medium without serum, and 50 ug/ml of the peptideof SEQ ID NO: 6, plus 2.5 ug/ml of human β2 microglobulin. The cellswere incubated in this mixture for four hours at 37° C., and were thenirradiated at 50 Gy. The cells were then centrifuged again, andresuspended in Iscove's medium and 10% human serum+AAG. The cells werethen placed in individual wells of 24 well tissue culture plates, at 1million cells per well.

Responder cells were then added. These were CD8⁺ T cells also obtainedfrom donor LB705. Fractions of CD8⁺ cells had been secured from thedonors' PBLs using well known techniques for separating T cellfractions. The responder cells were added to the wells, at 5×10⁶cells/well. Final volume was 2 ml. Following the addition of the cells,1000 U/ml of recombinant human IL-6 ("r-hu-IL-6"), and 10 ng/ml ofrecombinant human IL-12 ("r-hu-IL-12") were added.

Seven days later, i.e., on day 13, the responder cells, i.e., the CD8⁺cells, were restimulated. This was accomplished by transferring themixed culture discussed above to autologous adherent cells, togetherwith 10 U/ml r-hu-IL-2, and 5 ng/ml of r-hu-IL-7. The autologousadherent cells had been prepared previously, by incubating 5×10⁶irradiated (50 Gy) PBLs from LB705, in 1 ml Iscove's medium+10% humanserum+AAG, at 37° C. for two hours. Any non-adherent cells were removedand 50 ug/ml of the peptide of SEQ ID NO: 6 and 2.5 ug/ml of human β2microglobulin were added, in 0.5 ml of serum free medium. This mixturewas incubated for two hours at 37° C., and then washed. The responderCD8³⁰ cells were then added to them.

At day 21, another stimulation of the responder cells took place, byadding 2×10⁶ PBLs, irradiated at 50 Gy, which had been incubated inserum free medium+50 ug/ml human β2 microglobulin plus 50 ug/ml of SEQID NO: 6 for two hours, followed by washing.

This protocol resulted in the generation of CTLs specific for complexesof SEQ ID NO: 6 and HLA-A2, which is shown in the following example.

EXAMPLE 4

Experiments were carried out on day 28 to determine if peptides inaccordance with the invention, when complexed to HLA-A2 molecules, wouldprovoke lysis by CTLs.

Cells of line T2 were used. This cell line presents HLA-A2 molecules onits surface, but has an antigen processing defect which results inincreased capacity for presenting exogenous peptides. See Cerundolo, etal, Nature 345: 449 (1990), which describes this cell line. Otherequivalent cell lines are also available.

Samples of T2 cells were labelled with radioactive chromium (⁵¹ Cr), andincubated together with 1 μm of the peptide of SEQ ID NO: 6. Thepreincubation took place for one hour prior to introduction of CTLs.Control samples of T2 cells were not incubated with peptide.

CTLs were prepared by stimulating CD8⁺ cells with autologous APCs,preincubated with the peptide of SEQ ID NO: 6 for a period of 21 days,in accordance with example 3, supra.

FIG. 3 shows the results. The X-axis shows the ratio of effector/targetcells, while the Y axis depicts the percent of specific lysis,determined by measuring chromium release, in accordance with Boon, etal., J. Exp. Med. 152: 1184 (1980), incorporated by reference in itsentirety. In each test well, non-specific lysis was eliminated by adding50,000 K562 cells to the 1,000 ⁵¹ Cr labelled T2 target cells employed.The K562 cells act to eliminate non-specific lysis, as Natural Killer,or "NK" cells preferentially lyse this line.

It is clear that the peptide, when presented by HLA-A2, provoked lysisof the T2 cells.

EXAMPLE 5

The work described in example 4 led to the generation of a mixed cultureof CD8⁺ T cells specific for complexes of SEQ ID NO: 6 and itspresenting HLA-A2 molecules, and non-specific CTLs. To isolate CTLclones of the desired specificity, a limiting dilution assay was carriedout in accordance with Herin, et al., Int. J. Cancer 39: 390-396 (1987),incorporated by reference, but summarized herein.

On day 29, following Herin, et al., supra, irradiated SK23-MEL cells,known to express MAGE-3 and HLA-A2, were combined with the CTL mixedculture, together with LG2-EBV cells, which acted as feeder cells. Also,50 U/ml of IL-2, and 5 U/ml IL-4 were added to the mixture. Thisresulted in the generation of CTL clones 297/19, CTL 297/22, CTL 297/27,and CTL 297/36.

EXAMPLE 6

In example 4, the induction of lysis of cells presenting the peptide ofSEQ ID NO: 6 was shown, using a fixed amount of peptide with varyingeffector/target ratios. In these experiments, the effector/target ratiowas kept constant, and the amount of peptide varied. Again, the ⁵¹ Crrelease assay of example 4 was used, as were the T2 cells and CTLs ofexample 4. The four different CTLs of example 5 were used.

FIGS. 4A, 4B, 4C and 4D show these results, and indicate some dosedependency of lysis. When T2 cells were not incubated with peptide,lysis was always below 2%.

EXAMPLE 7

In another set of experiments, the lytic effect of the peptide of SEQ IDNO: 6 was tested in a model where the host cells did not inherentlyexpress HLA-A2.

Samples of cell line COS-7 were used. The cells were transfected withone of (i) genomic DNA for HLA-A2.01 and cDNA for MAGE-3, (ii) genomicDNA for HLA-A2.01 only, or (iii) cDNA for MAGE-3 only. In each case, thetransfecting vector was pcDNA/AmpI, where the DNA was ligated to EcoRIadaptors, and cloned into the EcoRI site of the plasmid in accordancewith manufacturer's instructions. The recipient cells were seeded, at15,000 cells/well into tissue culture flat bottom microwells inDulbecco's modified Eagles Medium ("DMEM") supplemented with 10% fetalcalf serum. Cells were incubated overnight, and medium was removed andreplaced by 30 ul/well of DMEM containing 10% Nu serum, 400 ug/ml DEAEdextran, 100 μM chloroquine, and 100 ng of the plasmids made asdescribed herein. As a further control, COS-7 cells transfected withHLA-A2 alone (via HLA-A2.01 in pcDNA/AmpI), were preincubated for onehour with 1 μM of the peptide of SEQ ID NO: 6, following example 4,supra.

In addition, melanoma cell lines which were known to express MAGE-3 andwhich were HLA-A2⁺ were used. These cell lines are identified in thefigures as LB 373, LB43, LB24 clone 409, and SK23. Cell line MZ2-MEL.43is HLA-A2⁻. In additional tests the line was also transfected with theHLA-A2 gene in pcDNA/AmpI.

A TNF release assay was used, following Traversari, et al,Immunogenetics 35: 145-152 (1992), incorporated by reference herein, butoutlined below, together with the modifications thereto. Specifically,1500 CTLs were combined with 30,000 target cells (CTLs were one of theclones discussed supra). The cells were cultured together, in thepresence of 25 u/ml of IL-2. Twenty-four hours later, supernatants fromthe cultures were tested against WEHI 164 clone 13 cells, which aresensitive to TNF. Sensitivity was increased by adding LiCl to the WEHI164 clone 13 cells (20 mM), in accordance with Beyart, at al, PNAS 86:9494-9498 (1989).

The results of these experiments are depicted in FIGS. 5A-5D. Eachfigure presents TNF release (pg/ml), for a different CTL clone, fortransfectants (top panel, each figure), and tumor cell lines (bottompanel). The figures show that MAGE-3 transfection alone is insufficientto provoke lysis, nor is HLA-A2 transfection. Transfection with bothMAGE-3 and HLA-A2 was sufficient, which is not surprising. What isunexpected, however, is the increase in lysis secured when peptide SEQID NO: 6 is added to cells transfected with HLA-A2 alone. Note thispattern in the second panel as well, where the data for"MZ2-MEL.43/HLA-A2+1 μM SEQ ID NO: 6" demonstrated superior lysis to allothers. These patterns, as indicated, are repeated over all CTL clonestested.

EXAMPLE 8

An additional set of experiments were carried out to test the lyticaffect of the peptide in a chromium release assay on tumor cells. TheTNF assay of example 6, supra, is more sensitive than a chromium releaseassay, so the latter would confirm results of the TNF assay.

The nature of the ⁵¹ Cr release assay was described supra, withreference to Boon, et al J. Exp. Med 152: 1184-1193 (1980). Using thesame assay, CTL clones 297/19 and 297/22 were used, with a series oftarget cells known to be HLA-A2 positive. The cell lines LB43 and SK23are also known to express MAGE-3. Cell line T2 is HLA-A2⁺, and MAGE-3⁻.

Even at low E/T ratios, there was significant lysis, showing the abilityto induce lysis.

The foregoing describes the identification of peptides derived from theMAGE-3 tumor rejection antigen precursor which interact with MHC class Imolecule HLA-A2. Of particular interest, and a part of the subjectmatter of the present invention, are the peptides represented by SEQ IDNOS: 1-10. These peptides are easily synthesized by Merrifield Synthesisor other peptide synthesis methodologies.

Of special interest are peptides which satisfy the following formulas:##STR1## where n is 4 or 5, and Xaa is any amino acid. Especiallypreferred are peptides such as SEQ ID NO: 6, which is exemplary of thisclass.

The peptides, as indicated, complex with HLA-A2 molecules, and thesecomplexes have been immunoprecipitated, thus leading to another featureof the invention, which is isolated complexes of the HLA-A2 molecule andeither one of these peptides.

Both the peptides and the complexes are useful in various ways. As wasshown, the peptides bind to the HLA-A2 molecule, and thus they areuseful in assays to determine whether or not HLA-A2 presenting cells arepresent in a sample. The peptide is contacted to the sample of interestin some determinable form, such as a labelled peptide (radiolabel,chromophoric label, and so forth), or bound to a solid phase, such as acolumn or agarose or SEPHAROSE bead, and the binding of cells theretodetermined, using standard analytical methods.

Both the peptides and the isolated complexes may be used in thegeneration of monoclonal antibodies or cytolytic T cell clones specificfor the aforementioned complexes. Those skilled in the art are veryfamiliar with the methodologies necessary to accomplish this, and thegeneration of a cytolytic T cell clone is exemplified supra. As cancercells present complexes of MAGE-3 derived peptides such as SEQ ID NOS:2, 6, 7, 8 and 10 and HLA-A2, these monoclonal antibodies and cytolyticT cells clones serve as reagents which are useful in diagnosing cancer.The chromium release assay discussed supra is exemplary of assays whichuse CTLs to determine targets of interest, and the art is quite familiarwith immunoassays and how to carry these out.

Cytolytic T cell clones thus derived are useful in therapeutic milieuxsuch as adoptive transfer. See Greenberg et al., J. Immunol. 136(5):1917 (1986); Reddel et al., Science 257: 238 (1992); Lynch et al., Eur.J. Immunol. 21: 1403 (1991); Kast et al., Cell 59: 603 (1989), all ofwhich are incorporated by reference herein. In this methodology, thepeptides set forth supra are combined with antigen presenting cells("APCs") to form stable complexes. Many such methodologies are known,for example, those disclosed in Leuscher et al., Nature 351: 72-74(1991); Romero et al., J. Exp. Med. 174: 603-612 (1991); Leuscher etal., J. Immunol. 148: 1003-1011 (1992); Romero et al., J. Immunol. 150:3825-3831 (1993); Romero et al., J. Exp. Med. 177: 1247-1256 (1993), andincorporated by reference herein. Following this, the presenting cellsare contacted to a source of cytolytic T cells to generate cytolytic Tcell clones specific for the complex of interest. Preferably, this isdone via the use of an autologous T cell clone found in, for example, ablood sample, taken from the patient to be treated with the CTLs. Oncethe CTLs are generated, these are reperfused into the subject to betreated in an amount sufficient to ameliorate the cancerous condition,such as inhibiting their proliferation, etcetera by lysing cancer cells.

Another aspect of the invention, shown in the examples, is the use ofthe combination of IL-6 and IL-12 to activate T cells, cytolytic T cellsin particular "Activation" as used herein, refers to the ability tocause the T cells to carry out their intended function. In the case ofCTLs, of course, this is the recognition and lysis of cells presentingon their surfaces appropriate combinations of peptide and MHC molecule.The activated T cells can then be used diagnostically, e.g., todetermine whether a particular peptide/MHC combination is present on acell subpopulation in a test sample. Also the use of the combinedcytokines can facilitate the identification of particular CTLs. It isknown that in a CTL sample, only a very small subpopulation is availablewhich is specific to an MHC/peptide combination. By using the cytokinesin combination with a sample presenting the desired combination, one candetermine activation, via lysis, and compare it to a control valueobtained where everything is kept constant except the sample is notmixed with the additional materials. This provides the requisite controlvalue.

Another feature of the invention is a kit useful in the activation of Tcells, the kit comprising in separate portions, interleukin-6 andinterleukin-12, the two separate portions being contained within acontainer means. The kits of interest may also include, e.g., a separateportion of peptide to be presented, and/or a vector or coding region foran MHC molecule, or even a vector or coding sequence which codes forboth the MHC molecule and the peptide. For example, the peptide may beSEQ ID NO: 6. The vectors may contain an HLA-A2 coding region or acombination of HLA-A2 and SEQ ID NO: 6. Another feature of the inventionis a composition consisting essentially of IL-6 and IL-12, in amountssufficient to activate T cells, such as CTLs.

"IL-6" and "IL-12" as used herein refer to all forms of these molecules,be they naturally occurring or produced recombinantly, human, murine, orany other species, as well as all variations of the molecule which havethe same activating properties of IL-6 and IL-12.

The amount of IL-6 and IL-12 used may vary; however, it is preferred touse from about 500 to about 1000 u/ml of IL-6, and from about 1 to about10 ng/ml of IL-12, although these ranges may vary, in accordance withthe artisan's findings.

Other aspects of the invention will be clear to the skilled artisan andneed not be reiterated here.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 13                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GlyLeuGluAlaArgGlyGluAlaLeu                                                   (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      AlaLeuSerArgLysValAlaGluLeu                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CysLeuGlyLeuSerTyrAspGlyLeu                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      IleLeuGlyAspProLysLysLeuLeu                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      HisLeuTyrIlePheAlaThrCysLeu                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      PheLeuTrpGlyProArgAlaLeuVal                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acid residues                                            (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      ThrLeuValGluValThrLeuGlyGluVal                                                510                                                                           (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acid residues                                            (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      AlaLeuSerArgLysValAlaGluLeuVal                                                510                                                                           (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acid residues                                            (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      LeuLeuLysTyrArgAlaArgGluProVal                                                510                                                                           (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acid residues                                            (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     AlaLeuValGluThrSerTyrValLysVal                                                510                                                                           (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     GlyIleLeuGlyPheValPheThrLeu                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The third Xaa is any 4 or 5                            amino acids                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     XaaLeuXaaGlyXaaLeu                                                            5                                                                             (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acid residues                                             (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (D) OTHER INFORMATION: The third Xaa is any 4 or 5                            amino acids                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     XaaLeuXaaGlyXaaVal                                                            5                                                                             __________________________________________________________________________

We claim:
 1. Isolated cytolytic T cell clone specific for a complex ofHLA-A2 and a peptide selected from the group consisting of: SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:
 10. 2. Theisolated cytolytic T cell clone of claim 1, wherein said peptide is SEQID NO:
 6. 3. A method for treating a subject with a cancerous conditioncharacterized by cancer cells which present complexes of HLA-A2molecules and a peptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10,comprising administering an amount of the isolated cytolytic T cellclone of claim 1 to said subject sufficient to lyse cancer cells.
 4. Themethod of claim 3, wherein said peptide is SEQ ID NO:
 6. 5. The methodof claim 3, wherein said cytolytic T cell clone is derived fromautologous cytolytic T cells.
 6. A method for identifying a samplecontaining a complex of HLA-A2 and a peptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO:
 7. SEQ ID NO: 8 andSEQ ID NO: 10 comprising:(a) contacting an isolated cytolytic T cell ofclaim 1 with a sample suspected of containing said HLA-A2-peptidecomplex under conditions sufficient for interaction of said cytolytic Tcell with said complex, and (b) detecting interaction of said cytolyticT cell with said HLA-A2-peptide complex.